A kind of separation and culture method of Dendrobium officinale suspension cells
A technology of dendrobium candidum and suspension cells, which is applied in the field of plant cell engineering, can solve problems such as the gap between supply and demand, low technical content in product development, and lack of quality standard system
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Embodiment 1
[0024] (1) Disinfection: ① Rinse the newborn root tip of Dendrobium candidum with tap water for 30 minutes, place the washed tissue in a sterilized flask that is too heavy for ultra-clean work and sterilize the surface with 75% ethanol for 1 minute, and then sterilize the tissue with sterilized Rinse with distilled water for 3 to 5 times. ② Then disinfect the tissue with 0.5% to 10% sodium hypochlorite for 5 to 10 minutes, discard the disinfectant, and rinse the tissue with sterilized distilled water for 3 to 5 times. ③ Disinfect again with 0.5% to 10% sodium hypochlorite for 3 to 5 minutes, discard the disinfectant, and rinse the tissue with sterilized distilled water for 3 to 5 times.
[0025] (2) Anti-browning: put the sterilized roots of Dendrobium officinale in the browning inhibition medium (Table 1) containing antioxidants, and then shake the flask for about 30 minutes to 1 hour. Then, remove moisture from the tissue using sterile filter paper.
[0026] (3) Separation...
Embodiment 2
[0031] (1) Disinfection: same as embodiment 1 step
[0032](2) anti-browning: same as embodiment 1 step
[0033] (3) Separation of explant cells and improvement of cell telomerase activity: put the root tip of Dendrobium officinale into a sterilized dish containing cutting fluid (Table 2) with antioxidant activity, remove the root cap, and cut from the incision About 1mm as explants, inoculated into WPM pre-medium and cultivated for 30min. Then the tissue was subjected to ultrasonic treatment for 10 min, and the ultrasonic frequency was 35 kHz, so as to increase the activity of tissue cell telomerase. The obtained tissue was inoculated into MS as the basic medium (1% agar, 3% sucrose, 5 mg / L NAA, 0.5 mg / L KT, pH 5.8) for callus induction. Dark cultivation was carried out during the induction process, the cultivation temperature was 25°C±1°C, and the air humidity was 75%±5%.
[0034] (4) Establishment of suspension cell line: After 35 days of culture, the cells with high mer...
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